Chapter 21
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Redox Properties of Proanthocyanidins and Their Health Implications 1
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J. L. Torres , L. Juliá , A. Carreras , S. Touriño , D. Lizárraga , C. Matito , I. Medina , and M. Cascante 2
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IIQAB-CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain University of Barcelona, MartíI Franquès 1, 08028-Barcelona, Spain IIM-CSIC, Eduardo Cabello 6, 36028-Vigo, Spain
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Plant polyphenols are appreciated as dietary supplements and functional food ingredients. Their putative health promoting action may originate, at least in part, from their influence on the delicate redox balance governing cell functions. In vivo, flavonoids, particularly catechins, may be antioxidants or prooxidants depending on their structure and concentration. The accurate evaluation of the redox behavior of flavonoids should help defining their beneficial or toxic effects. Proanthocyanidin (oligomeric catechin) fractions and bio-based products from grape, pine and witch hazel with variable proportions of catechol and pyrogallol (two and three adjacent phenolic hydroxyl groups respectively) moieties were prepared and tested on stable free radicals and for in vitro activity on skin and colon cells to show that there may be a relationship between the electron transfer capacity of proanthocyanidins and their influence on cell functions such as proliferation and apoptosis.
Polyphenols of plant origin present in foods and supplements (1) are appreciated as antioxidant chemopreventive agents against a variety of diseases in which oxidative stress plays a significant role (2-4) although no conclusive proof of this beneficial action has ever been provided (5, 6). The primary
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© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
239 beneficial role of polyphenols is believed to be the scavenging of harmful reactive oxygen species (ROS) generated by the mitochondria. Flavanols are particularly efficient scavengers due to the presence of polyphenolic structures such as catechol, pyrogallol and gallate moieties (Figure 1). Moreover it is becoming evident that the biological effects of polyphenols involve other mechanisms in which the redox potential also appears to play a crucial role. Interestingly, the same structures may be antioxidant and pro-oxidant (see Figure 2 for a simplified model) depending on the biological microenviron-ment. For instance, pyrogallol is able to scavenge the superoxide radical and also to form it from molecular oxygen (7). The superoxide radical may trigger the formation harmful species such as the hydroxyl radical via the Fenton reaction (8) and peroxynitrites via nitric oxide (9), among others. In the particular case of apoptosis (programmed cell death) superoxide and oxidative stress appears to be determinant for triggering or halting the cascade of events (10). Whether the antioxidant/pro-oxidant effects of catechins are preventive, therapeutic or toxic will depend on where, how and how much the delicate redox balance of the aerobic organism is altered (77). These actions will be significant only in organs where polyphenols are bioavailable after oral administration, namely the gastrointestinal tract and particularly the colon.
Electron Transfer and Superoxide Anion Not all phenolic groups in catechins are equally reactive in terms of superoxide formation and pro-oxidant capacity. The pyrogallol moiety (three hydroxyls) of catechins such as (-)-epigal!ocatechin and (-)-epigallocatechingallate appear to be the most active pro-oxidant structure (72, 13) whereas the catechol moiety (two hydroxyls) of (-)-epicatechin barely participates in this so called redox cycling (14) and functions only as antioxidant scavenger. Because the superoxide anion is formed by transferring an electron to molecular oxygen (Figure 2) it appears that the high electron transfer capacity of pyrogallol compared to catechol is, at least in part, responsible for the prooxidant action of some catechins. To estimate the scavenging efficiency of catechins and, most important, their possible pro-oxidant and/or pro-apoptotic activities electron transfer capacity is an important parameter to measure.
Measurement of Electron Transfer Capacity and Apoptosis in Colon Cells We have recently introduced a new stable radical (HNTTM, Figure 3) reactive only through electron transfer (75, 16). This can be used to evaluate the capacity of catechins to donate electrons to ROS and to provide an estimation of
Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
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R= H, OH
ROS
Inactive ROS
R= H, OH
OH HO
OH
OH
''OH
r
''OH
OH
Figure 2. Scavenging of ROS and superoxide formation by catechins.
Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
241 their possible pro-oxidant and pro-apoptotic activities. We have tested a variety of catechin monomers, catechin conjugates with cysteamine and cysteine (17, 18) and proanthocyanidin fractions for their activity against HNTTM and compared the results with the apoptosis triggered by the same polyphenols on HT29 colon carcinoma cells. First the antiproliferative potency of polyphenols was determined and then apoptosis (early and late/necrosis) was assessed by FITC-FACS after treatment of the cells for 72 h at their respective IC50 concentrations. To assess the possible artifactual results due to autooxidation of the catechins (14) control experiments with iron free medium were conducted. The catechin-gallate monomers and conjugates triggered some early apoptosis (5-10% of total cells) and showed high electron transfer capacity values (6-7 electrons per molecule) while gallocatechin-gallates also showed high electron transfer values but less early apoptosis. In agreement with this the gallocatechins were the most efficient antiproliferative compounds on colon carcinoma cells. This, together with the late apoptosis and necrosis detected for gallocatechins might be associated with the pyrogallol group on ring B.
Figure 3. Tris(2,4,5-trichloro-3,5-dinitrophenyl)methyl radical (HNTTM).
We have also tested proanthocyanidin extracts and fractions with variable proportions of gallocatechins and gallates. Table I summarizes the results obtained with some significant fractions from pine (no gallocatechins, no gallates), witch hazel (high gallocatechin and gallate content) and grape (intermediate gallocatechin and gallate content). The electron transfer capacity and apoptosis were estimated as stated before and the structural analysis, including the estimation of the mean molecular weight used to calculate the mean electron transfer capacity was done by thioacidolysis with cysteamine as described (19). In agreement with the conclusions on monomers, the results
Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
242 show that apoptosis and particularly late apoptosis and necrosis may be due to the presence of pyrogallol moieties (gallocatechins) in the fractions with high electron transfer capacities.
Table I. Electron Transfer capacity and Induction of Apoptosis by Selected Fractions b
Composition" Electron transfer
Apoptosis'
Fraction IV Pine IV Grape IV Witch Hazel
0,0
